Composition containing monoacetyldiacylglycerol compound as active ingredient for inhibiting blood cancer or metastasis

ABSTRACT

The present invention relates to a composition containing a monoacetyldiglyceride compound as an active ingredient, for inhibiting blood cancer or metastasis, and a use thereof. The monoacetyldiglyceride compound according to the present invention has excellent effects of inhibiting the expression of IL-4 and inhibiting the activity of STAT-6, and thereby is capable of overcoming side effects of currently used blood cancer or metastasis inhibiting agents. Also, the monoacetyldiglyceride compound is a non-toxic compound having superior therapeutic effects and thus can be useful as a composition for preventing, treating, or improving blood cancer and metastasis.

TECHNICAL FIELD

The present invention relates to a composition for inhibiting bloodcancer or metastasis, comprising a monoacetyldiglyceride compound as anactive ingredient, and a use thereof.

BACKGROUND ART

Cancer, which is also referred to as a tumor, means groups of abnormalcells which are generated by unlimitedly continued fragmentation andproliferation of cells, originating from destruction of the balancebetween cell division and apoptosis by a variety of causes. In general,cancers are expressed in various parts of the body more than 100 parts,including organs, white blood cells, bones, lymph nodes, etc, and thenare developed into severe symptom through infiltration into thesurrounding tissue and transition to a different organ. The causes ofcancer include environmental or external factors such as the chemical,viral, bacterial, ionizing radiation and so on and internal factors suchas congenital gene mutations and so on. Interest in the relation betweenchronic inflammation and cancer has been recently newly appeared andmany data demonstrating the relation between the chronic inflammationand cancer has been reported. Infection and chronic inflammation occupy25% of all cancer cause and it has been known that cancer risk is muchhigher in patients having chronic inflammation and ROS (Reactive OxygenSpecies)-associated diseases. It is speculated that a variety ofmediators that regulate the inflammatory response, that is, cytokine,free-radical, growth factor, etc. induce genetic, epigenetic changessuch as mutations in tumor suppressor genes, DNA methylation,post-transcriptional modification, etc., change the route essential formaintaining cell homeostasis, and further generate and develop cancer.

If cancers are found in the initial, medical treatment such as radiationtherapy or chemotherapy etc can be applied to the patients. However, theside effect of the medical treatment becomes a big problem. If cancersare found in the terminal or in the metastasis state, the patientsshould dwell in life-limiting condition without special treatment.Therefore, the research has been conducted for developing anti-canceragent or cancer metastasis inhibitor having less side effect and greatefficacy from low-toxic natural products, as a new approach for thecancer treatment. It had been found that the treatment originating inthe natural products reduces the side effect of the suppression ofhematopoiesis and immune function which is often observed inchemotherapy and radiation therapy.

EC-18, as a kind of monoacetyldiglyceride compounds, was separated orextracted from the natural deer antler. It is known that EC-18 increasessurvivability ratio of animals in sepsis animal model experiment usingcecal-ligation-puncture, and shows no-toxicity in GLP (Good LaboratoryPractice) toxicity test. However, the effect of monoacetyldiacylglycerolcompounds including EC-18 is not known or disclosed in the blood canceror cancer metastasis. Thus the present inventors aimed to find acompound derived from natural products or a novel compound for theinhibitor of blood cancer or cancer metastasis and found that themonoacetyldiacylglycerol compound inhibits IL-4 secretion and STAT-6activation to destroy micro-environment for the growth of the cancertissue and can be used to prevent or treat blood cancer or inhibitcancer metastasis.

DISCLOSURE Technical Problem

It is an object of the present invention to provide a pharmaceuticalcomposition and a health functional food containing amonoacetyldiacylglycerol compound represented by following Formula 1 asan active ingredient for inhibiting blood cancer or cancer metastasis.

wherein R1 and R2 are independently a fatty acid residue of 14 to 22carbon atoms.

It is an object of the present invention to provide a method forpreventing or treating blood cancer or a method for inhibiting cancermetastasis comprising a step of administering the above composition to asubject suspicious of having blood cancer attack or cancer metastasis,or suffering from blood cancer.

Technical Solution

In an example to achieve the these and other objects, the presentinvention provides a pharmaceutical composition and a health functionalfood containing a monoacetyldiacylglycerol compound represented byfollowing Formula 1 as an active ingredient for inhibiting blood canceror cancer metastasis.

wherein R1 and R2 are independently a fatty acid group of 14 to 22carbon atoms. In the Specification, the fatty acid group means thecarboxyl group of fatty acids from which —OH group is extracted.

In detail, the pharmaceutical composition for inhibiting blood cancer orcancer metastasis according to the present invention includes amonoacetyldiacylglycerol compound represented by the Formula 1. In thepresent invention, the term “monoacetyl diacyl glycerol compound” meansglycerol compounds having one acetyl group and two acyl groups, and canbe referred as “monoacetyl diacyl glycerol (MADG)”.

In the monoacetyl diacyl glycerol compound of Formula 1, R1 and R2 areindependently a fatty acid residue of 14 to 22 carbon atoms. Preferably,non-limiting examples of R1 and R2 include palmitoyl, oleoyl, linoleoyl,linolenoyl, stearoyl, myristoyl, arachidonoyl, and so on. Preferablecombinations of R1 and R2 (R1/R2) include oleoyl/palmitoyl,palmitoyl/oleoyl, palmitoyl/linoleoyl, palmitoyl/linolenoyl,palmitoyl/arachidonoyl, palmitoyl/stearoyl, palmitoyl/palmitoyl,oleoyl/stearoyl, linoleoyl/palmitoyl, linoleoyl/stearoyl,stearoyl/linoleoyl, stearoyl/oleoyl, myristoyl/linoleoyl,myristoyl/oleoyl, and so on. In optical activity, the monoacetyl diacylglycerol compound of Formula 1 can be (R)-form, (S)-form or a racemicmixture.

In one embodiment, the monoacetyl diacyl glycerol compound is a compoundof the following Formula 2.

The compound of Formula 2 is 1-palmitoyl-2-linoleoyl-3-acetylglycerol,sometimes referred as “EC-18” in this specification. R1 and R2 of thecompound of Formula 2 are palmitoyl and linoleoyl, respectively.

The monoacetyldiacylglycerol compounds can be separated and extractedfrom the natural deer antler or can be produced by known organicsynthesis methods (Korean Registered Patents No. 10-0789323). Morespecifically, deer antler is extracted with hexane, followed byextracting the residue with chloroform and removing the chloroform toprovide chloroform extracts. The volume of the solvents for thisextraction is just enough to immerse the deer antler. In general, about4-5 liters of hexane and/or chloroform for 1 kg of deer antler is used,but not limited thereto. The extracts obtained by this method is furtherfractionated and purified using series of silica gel columnchromatograph and TLC method to obtain the monoacetyldiacylglycerolcompound for the present invention. A solvent for the extraction isselected among chloroform/methanol, hexane/ethylacetate/acetic acid, butnot limited thereto.

A chemical synthetic method for the preparation ofmonoacetyldiacylglycerol compounds is shown in Korean Registered PatentsNo. 10-0789323. Specifically, the method comprises (a) a step ofpreparing 1-R1-3-protecting group-glycerol by adding a protecting groupin the position 3 of 1-R1-glycerol; (b) a step of preparing1-R1-2-R2-3-protecting group-glycerol by introducing R2 in the position2 of the 1-R1-3-protecting group-glycerol; and (c) a step of preparingthe desired monoacetyldiacylglycerol compound by performing adeprotection reaction and the acetylation reaction of the1-R1-3-protecting group-glycerol at the same time. Themonoacetyldiacylglycerol compound may be further purified if necessary.Alternatively, monoacetyldiacylglycerol compounds can be prepared byacid decomposition of phosphatidylcholine (acetolysis) but is notlimited thereto. Stereoisomers of the compounds of formula (I) are alsowithin the scope of the invention.

In the present invention it is shown that monoacetyldiacylglycerolcompounds are capable of reducing the secretion of IL-4, and thus themonoacetyldiacylglycerol compounds can be effectively used for thesuppression of the blood cancer or the cancer metastasis. \

In the present invention, the term “cancer” indicates a state that theabnormal cells which should be normally killed but alive and excessivelyproliferated by control disorder of cell itself, invade surroundingtissues and organs, to form lumps and destroy or modify the existingtissue and/or organic structure, and the cancer is used interchangeablywith the malignancy. The term “anticancer” means all the activity forinhibiting proliferation of cancer cells or killing the cancer cells. Inthe present invention, the “blood cancer” is one selected from a groupconsisting of lymphoma, acute leukemia, chronic leukemia and multiplemyeloma, but are not limited thereto. The term “metastasis” means thespread of cancer cells, especially cancer cells associated with bloodcancer, from one organ or part thereof to surroundings thereof oranother organs, but is not limited thereto. Malignant cancer cellsmainly have the ability for metastasis. Cancer cells exit from a primarycancer to the lymph system or blood system, circulate the blood vesselsand grow in the normal tissues at other parts of the body. Cancermetastasis, as a typical feature of malignant cancer, is accounted for90% of deaths due to cancer. Therefore, cancer metastasis inhibition inthe present invention is used as the means for inhibiting the spread ofcancer cells to other organs or the surroundings. In the presentinvention, the term “prevention” means any activities to suppress ordelay onset of cancer or cancer metastasis by the administration of thepharmaceutical composition of the present invention and “treatment”means any action to improve symptoms caused by the cancer and/or cancermetastatic or to change symptoms by the cancer and/or cancer metastaticto more beneficial states.

Tumor-Associated Macrophage (TAM), as macrophages associated with tumorprogression and metastasis, is found mostly in periphery of tumor. Amethod of improving the tumor microenvironment in a way to attack theTAM by immunizing molecules overexpressed by the TAM has been emerged asa new anti-cancer therapy. The TAM, as a M2 macrophage phenotype, hasbeen primarily known to be induced by Th2 cytokines such as IL-4, IL-13and so on. Actually the TAM secretes angiogenesis stimulating factor andmetalloproteinase, and is involved in the signaling pathways thatcontrol the action of the tumor stromal fibroblasts to promoteproliferation and metastasis of tumor cells.

In the present invention, the term “Interleukin-4 (IL-4)” meanscytokines having various immunomodulatory functions secreted from Th2lymphocytes, eosinophils, mast cells, etc. It has been reported that theIL-4 is found at a higher concentration than normal tissue, in manycancer cells and a large amount of IL-4 is produced intumor-infiltrating lymphocytes (TILs). Especially, the IL-4 has beenknown to a representative cytokine which activates the TAM. Accordingly,the IL-4 induces the activation of the M2 macrophage phenotype M2 andthus induces tumor growth, metastasis, angiogenesis and so on.

Also, it is shown that monoacetyldiacylglycerol compounds are capable ofinhibiting the activation of STAT-6, and thus themonoacetyldiacylglycerol compounds can be effectively used for thesuppression of the blood cancer or the cancer metastasis. The term“STAT-6” of the present invention, as a transcription factor, has beenknown to play an important role for performing IL-4-mediated Biologicalresponse. That is, the activation of STAT6 in the form phosphorylated byIL-4 is followed by the activation of IL-4/STAT-6 signal pathway. Thesignaling pathway has been known to play an important role in resistanceto the cell proliferation/growth and apoptosis. Accordingly, thesuppression of the STAT-6 makes an induction of apoptosis and aninhibition of metastasis, and a destruction of the micro-environment forthe growth of the cancer tissue, so that the composition of the presentinvention can be used for remedy and remedy combination effective in thetumor.

In the examples of the present invention, it is shown that i) when U937cell, A549 cell and Jurkat cell each is treated with IL-4 and EC-18, thephosphorylation of STAT6 is inhibited depending on concentration ofEC-18(Experimental Example 1, FIG. 1 and FIG. 2), and ii) when STAT-6 istreated with EC-18 after activating STAT-6 by treating HEK293 cell andA549 cell with IL-4, the activity of STAT-6 is reduced(ExperimentalExample 2, FIG. 3). In another example of the present invention, it wasshown that the secretion amount of IL-4 is reduced depending uponconcentration of EC-18, from the observation on the transfer amount ofIL-4 in accordance with the concentrations of the EC-18 by enzyme-linkedimmunosorbent absorption analysis (ELISA), in RBL −2H3 cell(Experimental Example 3, FIG. 4 and FIG. 5), which suggests themonoacetyldiacylglycerol compounds are effective in the treatment ofblood cancer or metastasis. In detail, RPMI 8226 cell of bone cancercell line originating in human body is transplanted into male nude miceand EC-18 of the test substance is administered orally, thereafter thesuppression effect of tumor growth is evaluated. At a test substanceadministration group of 500 mg/kg dose, the tumor volume isstatistically significantly decreased, and also weight of the tumor isstatistically significantly decreased, as compared to negative controlgroup. Accordingly it is shown that EC-18 suppress the tumor growth(Experimental Example 4, FIG. 6 and FIG. 7), which demonstrates thatmonoacetyldiacylglycerol compounds are effective for the prevention ortreatment of the blood cancer or inhibition of cancer metastasis.

The pharmaceutical composition containing monoacetyldiacylglycerolcompounds of the present invention may additionally include conventionalpharmaceutically acceptable carriers, excipients, or diluents. Theamount of monoacetyldiacylglycerol compounds in the pharmaceuticalcomposition can be widely varied without specific limitation, and isspecifically 0.0001 to 100.0 weight %, preferably 0.001 to 50 weight %,more preferably 0.01 to 20 weight % with respect to the total amount ofthe composition.

The pharmaceutical composition may be formulated into various forms fororal or non-oral administration, for example one selected from a groupconsisting of tablet, bolus, powder, granule, capsule such as hard orsoft gelatin capsule, emulsion, suspension, syrup, emulsifiableconcentrate, sterilized aqueous solution, non-aqueous solution,freeze-dried formulation, suppository, and so on. In formulating thecomposition, conventional excipients or diluents such as filler, bulkingagent, binder, wetting agent, disintegrating agent, and surfactant canbe used. The solid formulation for oral administration includes tablet,bolus, powder, granule, capsule and so on, and the solid formulation canbe prepared by mixing one or more of the active components and at leastone excipient such as starch, calcium carbonate, sucrose, lactose,gelatin, and so on. Besides the excipient, a lubricant such as Magnesiumstearate and talc can also be used. The liquid formulation for oraladministration includes emulsion, suspension, syrup, and so on, and mayinclude conventional diluents such as water and liquid paraffin or mayinclude various such as wetting agent, sweeting agent, flavoring agent,and preserving agent. The formulation for non-oral administrationincludes sterilized aqueous solution, non-aqueous solution, freeze-driedformulation, suppository, and so on, and solvent for such solution mayinclude propylene glycol, polyethylene glycol, vegetable oil such asolive oil, and ester for syringe injection such as ethyl oleate. Basematerials of the suppository may include witepsol, macrogol, tween 61,cacao butter, Laurin and glycerogelatine.

The composition of the present invention can be administered in apharmaceutically effective amount. The term “pharmaceutically effectiveamount” is used to refer to an amount which is sufficient to achieve adesired result in a medical treatment. The “pharmaceutically effectiveamount” can be determined in accordance with type, age and sex of asubject, severity and type of disease, activity of drug, sensitivity todrug, administration time, period and route, excretion rate, and otherwell known criteria in medical field. The composition of the presentinvention can be administered alone or with other medicines sequentiallyor simultaneously, or administered once or several times. Consideringall the above factors, it is important to dose the amount that canachieve the maximum effect with the minimum amount with no side effects,which can be readily determined by those skilled in the art. Thepreferable amount of the composition of the present invention can bevaried in accordance with the condition and weight of patient, severityof disease, formulation type of drug, administration route and period ofdrug. Appropriate total amount of administration per 1 day can bedetermined by a doctor of related medical filed, and generally 0.001 to1000 mg/kg, preferably 0.05 to 200 mg/kg, more preferably 0.1 to 100mg/kg once or several times by dividing in 1 day. The composition of thepresent invention can be administered to any subject which requires thesuppression of blood cancer or cancer metastasis. For example, thecomposition of the present invention can be administered to not onlyhuman but also non-human animal (specifically mammals) such as monkey,dog, cat, rabbit, guinea pig, rat, mouse, cow, sheep, pig, goat, and soon. The composition of the present invention can be administered byconventional various methods, for example, by oral or rectumadministration, or by intravenous, intramuscular, subcutaneous orcerebrovascular injection.

As other aspect of the present invention, the present invention providesa health functional food for inhibiting blood cancer or cancermetastasis comprising monoacetyldiacylglycerol compounds of Formula 1 asan active component(s),

wherein R1 and R2 are independently a fatty acid group of 14 to 22carbon atoms, but are not limited thereto.

In detail, the monoacetyldiacylglycerol compounds of the presentinvention or pharmaceutically acceptable salt thereof can be included inthe health functional food for the suppression of blood cancer or cancermetastasis. The monoacetyldiacylglycerol compounds, suppression of bloodcancer or cancer metastasis are previously explained in detail. The term“improving” means every change which reduces or advantageously changesthe symptoms in a subject having or suspicious of having blood cancer orcancer metastasis.

When the composition of the present invention is included in the healthfunctional food, the composition can be included alone or with otheractive component. The amount of the compounds of the present inventionin the health functional food can be determined in accordance with theintended use of the health functional food. Generally, when preparinghealth functional food or beverage, the composition of the presentinvention can be included in the amount of less than 15 weight part,preferably less than 10 weight part. In case of long term administrationfor maintaining one's health, the amount of the composition can bereduced. However, since the active component does not cause any adverseeffect, the amount of the composition can be increased by more than theabove mentioned amount. The health functional food including thecomposition of the present invention can be any conventional food orbeverage. Specific examples of the food include meat, sausage, bread,chocolate, candy, snack, biscuit, pizza, Ramen, noodles, gum, ice cream,dairy product, soup, beverage, tea, drink, alcoholic drink, vitamincomplex, and so on. If necessary, the food of the present invention canalso include food for an animal.

When the health functional food is beverage, the beverage may includeconventional sweetener, flavoring agent, natural carbohydrate, and soon. Examples of the natural carbohydrate include monosaccharide such asglucose and fructose, disaccharide such as maltose and sucrose,polysaccharide such as dextrin and cyclodextrin, and sugar alcohol suchas xylitol, sorbitol, and erythritol. The preferable amount of thenatural carbohydrate can be about 0.01 to 0.04 g, more preferably about0.02 to 0.03 g with respect to 100 Ml of the beverage of the presentinvention. Examples of the sweetener includes natural sweeteners such asThaumatin and Stevia extract and artificial sweeteners such as saccharinand aspartame. The health functional food of the present invention mayfurther include various nutritional supplement, vitamin, electrolyte,flavoring agent, coloring agent, pectic acid and its salt, alginic acidand its salt, organic acid, protective colloid, thickener, pH adjuster,stabilizer, preservative, glycerin, alcohol, juice and so on.

As another aspect of the present invention, the present inventionprovides a method for preventing or treating blood cancer or forinhibiting cancer metastasis comprising a step of administering thepharmaceutical composition to a subject who is suspicious of havingblood cancer or cancer metastasis. The “subject who is suspicious ofhaving blood cancer or cancer metastasis” includes not only an animalincluding human being which has blood cancer or cancer metastasis butalso potentially has blood cancer or cancer metastasis. The subject whois suspicious of having blood cancer or cancer metastasis can beeffectively treated by administering the pharmaceutical composition ofthe present invention. The term “administering” means introducing thepharmaceutical composition of the present invention into the subject whois suspicious of having blood cancer or cancer metastasis by any means.The administration route can be any route such as oral or non-oralroute.

The method for treating blood cancer comprises a step of administeringan effective amount of a pharmaceutical composition comprising themonoacetyldiacylglycerol compounds of formula I or pharmaceuticallyacceptable salt thereof to a patient in need thereof. An appropriatetotal amount of administration per 1 day can be determined by aphysician, and is generally about 0.001 to about 1000 mg/kg, preferably,about 0.05 to 200 mg/kg, more preferably about 0.1 to about 100 mg/kg.The total administration amount per day can be administered once a dayor can be administered in divided doses multiple times daily. However,the specific therapeutically effective amount of themonoacetyldiacylglycerol administered to a particular patient can bevaried depending on the type and degree of the response to be achievedin the treatment, the specific composition, including whether anotheragent is included in the composition, the patient's age, body weight,general health status, sex, diet, administration time, administrationroute, the ratio of composition, treatment period, other drugs usedtogether in the treatment and a variety of factors well known in themedical field.

Effect of Invention

Monoacetyldiacylglycerol compounds of the invention are excellent in theeffect of inhibiting the expression of IL-4, and inhibiting the activityof STAT-6, thereby overcoming side-effects of currently used inhibitorfor blood cancer or cancer metastasis, while having no toxicity andexcellent treatment effect so that Monoacetyldiacylglycerol compoundscan be usefully used for the prevention, treatment and improvement ofblood cancer or cancer metastasis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents Western blot analysis in U937 cell, showingphosphorylation inhibitory effect of (A) STAT-6 which is phosphorylatedby IL-4, depending on concentration of EC-18 and (B) STAT-6 which isphosphorylated by IFN-γ, depending on concentration of EC-18.

FIG. 2 presents Western blot analysis in A549 cell(A), U937 cell(B) andJurkat cell(C), showing phosphorylation inhibitory effect of STAT-6which is phosphorylated by IL-4, depending on concentration of EC-18.

FIG. 3 presents the values of the light-emitting luciferase of STAT-6which is activated by IL and STAT-1 which is activated by IFN-γ,depending on concentration of EC-18, in HEK293 cell and A54 cell.

FIG. 4 presents the transfer amount of IL-4 depending on concentrationof EC-18, in RBL-2H3 cell, which was measured by an enzyme-linkedimmunosorbent absorption analysis (ELISA).

FIG. 5 shows the expression level of IL-4 over time (6 h, 15 h and 24 h)depending on concentration of EC-18, in RBL-2H3 cell.

FIG. 6 is a graph showing the reduction of tumor volume by the EC-18treatment in the model animal with which human blood cancer cell aretransplanted.

FIG. 7 is a graph showing the reduction of tumor weight by the EC-18treatment in the model animal with which human blood cancer cell aretransplanted.

FIG. 8 presents photographs showing the size change of the tumor, inEC-18 treated group, negative control and positive control of bloodcancer mouse model.

DETAILED DESCRIPTION OF THE INVENTION

A more detailed description of the invention will be made by referenceto the attached drawings, which are intended for better understanding ofthe present invention and will not limit the present invention.

Example: Cell Culture

The human cell line U937, A549, Jurkat, HEK293 and rats cell lineRBL-2H3 (American Type Culture Collection, ATCC, Rockville, Md.) werecultured at 37° C. under 5% CO₂ humid condition. U937, Jurkat, K562 andA549 cell lines were cultured in RPMI1640 (Life Technologies, Karlsruhe,Germany) medium containing 10% elegant serum (Fetal Calf Serum, FCS,HyClone, Logan, Utah), 2 mM L-glutamine, 100 μg/Ml penicillin and 100μg/Ml streptomycin (Life Technologies), HEK293 cell line was cultured inDMEM medium, and RBL-2H3 cell line was cultured in MEM medium.

Experimental Example 1: Inhibitory Effect of EC-18 on STAT-6Phosphorylation

Cells treated with EC-18 and IL-4 and IFN-γ were lysed with SDS-coldlysis buffer [(50 mM HEPES, 150 mM NaCl, 0.2 mM EDTA, 0.5% NP-40, 0.1%SDS, 1 mM Na₃VO₄, 10 mM NaF, and complete Protein Inhibitor Cocktail(Roche)] for 30 minutes on ice. After cell Subsequently, the RNA wasisolated after dissolving the cells, the aqueous solution was separatedfrom the insoluble precipitate by centrifuging the cell lysates for 30minutes at 13,000 rpm in a high-speed centrifuge. After quantifying theseparated cell aqueous solution, the quantified solution was separatedin 10 to 12% SDS-PAGE by electrophoresis. The cellular proteinsseparated in the Gel were transferred to PVDF membrane (Millipore,Billerica, Mass., USA) for two hours at 100V.

To measure the amount of phosphorylated STAT1, STAT6 in cells, themembrane was incubated with poly rabbit anti- (STAT1, STAT6), -phosphor(STAT1, STAT6) (Cell signaling Technology, USA) (1:1000) as a primaryantibody at room temperature for 60 minutes. The membrane was incubatedwith horseradish peroxidase peroxidase-conjugated goat anti-rabbit IgG(Santa Cruz Biotechnology, USA)(1:3000) as a secondary antibody at roomtemperature for 60 minutes. The same amount of cellular proteins wasconfirmed by poly rabbit anti- (STAT1, STATE) antibody. After incubationwith antibodies, membrane was incubated with ECL solution (Millipore,Billerica, Mass., USA) and exposed to X-ray film. The amount ofphosphorylated STAT was measured in the band on the film.

The result shows that STAT-6 is phosphorylated in A549 cells ofepithelial cells and U937 and Jurkat cells of immune cells, and theamount of phosphorylated STAT-6 is decreased by the pre-treatment ofEC-18 in a concentration-dependent manner (FIG. 1-A, FIGS. 2-A to C).FIG. 1B shows that there is no phosphorylation decrease at STAT-1 byEC-18.

Experimental Example 2:Measurement of Inhibitory Effect of EC-18 onSTAT-6 Activation Using the Luciferase Reporter

The p4xSTAT6-Luc2P vector (Addgene) containing the STAT-6 Element (SIE)that STAT-6 binds to was introduced into HEK293 cells and A549 cells.Inhibition of the STAT-6 activation by EC-18 was measured bypre-treating the cells with EC-18 and then treating the cells with IL-4.Also, the activation of STAT-1 was measured by usingpGL4.45[luc2P/ISRE/Hygro] vector (Promega) containing InterferonStimulated Response Element (ISRE) that STAT-1 binds to.

After disrupting cells by treatment with trypsin-EDTA, HEK293 cells andA549 cells were split to the culture plate. Using AttracteneTransfection Reagent (Qiagen), cells were transfected withp4xSTAT6-Luc2P and pGL4.45[luc2P/ISRE/Hygro] and and incubated at 37° C.under 5% CO₂ conditions for a day. In the next day, the cells werecollected from the culture plate and 0.1 ml of cells were transferred to96-well plate in a concentration of 5×10⁴ cells/well and incubated at37° C. under 5% CO₂ conditions for a day. In the next day, the cellswere pre-treated with different concentrations of EC-18 for one hour andthen treated with 10 ng/ml of IL-4 and IFN-γ. After incubation at 37° C.under 5% CO₂ conditions for 6 hours, Luciferease activity was measuredusing ONE-Glo Luciferase Assay System (Promega). Specifically, 0.1 ml ofa 1:1 mixture of ONE-Glo Luciferase Assay System and Substrate was addedto each well. After three minutes, the fluorescence by IL-4 and IFN-γwas measured ONE-Glo Luciferase Assay System (Promega) as follows.Specifically, 0.1 ml of a 1:1 mixture of ONEONE-Glo™ Luciferase AssayBuffer and Substrate was added to each of 96-well. After three minutes,the fluorescence was measured using VICTOR X Multilabel Plate Reader(PerkinElmer) during 0.5 second.

Luciferase fluorescence was decreased in cells pre-treated withdifferent concentrations of EC-18, compared to cells treated with IL-4alone, indicating decreased STAT-6 activity in cells pre-treated withEC-18. On the other hand, the Luciferase fluorescence of the STAT-1 byEC-18 was maintained in cells treated with IFN-γ (FIG. 3). The resultshows that STAT-6 activation induced by IL-4 is decreased by thetreatment of EC-18 in HEK293 cells and A549 cells.

Experimental Example 3: IL-4 Gene Expression Inhibitory Effect of theEC-18 Experimental Example 3-1: Reverse Transcriptase Polymerase ChainReaction (RT-PCR)

After pre-treatment of RBL-2H3 cells with EC-18, cellular activity wasinduced by antigen for IgE, and as a result, the change in the mRNAlevels of the cytokines IL-4 expressed in RT-PCR was performed (ReverseTranscriptase Polymerase Chain Reaction. Total RNA was isolatedaccording to standard protocols, cDNA was synthesized using theAccuScript High Fidelity 1st Strand cDNA Synthesis Kit (Stratagene)according to the manufacturer's instructions. Step 2 RT-PCR reactionswere performed using the oligo-dT primer and reverse transcriptase, aprimer pair specific and Taq polymerase (Takara, Shiga, Japan). cDNAsynthesis of 1 μl was used in PCR reaction of 20 μl constituting 0.5 UTaq DNA polymerase, 1 buffer and 1 mM dNTP mix (Takara) and specificprimer pairs.

Amplification by PCR was performed using a GeneAmp PCR system 2700(Applied Biosystems, Foster city, CA, USA) under the followingconditions: 5 minutes at 94°, then 45 seconds at 94°, 45 seconds at 56°and 1 minute at 72°, and then subjected to 25 to 40 cycles). Finalextension reaction was carried out at 72° for 7 minutes. Table 1 showsthe primer sequences used in the cDNA amplification by PCR. PCR primerswere designed using Primer3 program and were purchased from Bioneer,Inc. (Republic of Korea, Daejeon).

TABLE 1 Name Forward primer Reverse primer GAPDH CCATCACCATCTTCCAGGAGACAGTCTTCTGGGTGGCAGT (SEQ ID NO: 1) (SEQ ID NO: 2) IL-4AATGGGTCTCACCTCCCAAC TTCAGCTCGAACACTTTGAA (SEQ ID NO: 3) (SEQ ID NO: 4)

PCR product was separated on a 1.5% agarose gel, stained with ethidiumbromide to (ethidium bromide, EtBr), visualized by Gel Doc 2000 UVtrans-illuminator (Bio-Rad Laboratories, Hercules, Calif., USA), andthen analyzed by using Quantity One software (Bio-Rad Laboratories).Each sample was tested at least three times and data representative wasproposed. Cells were pre-treated with 0.01, 0.1, 1.0, and 10 μg/Ml ofEC-18 respectively for 1 hour and antigen was processed, and theincubated for 3 hours. Subsequently, the RNA was isolated after celllysis. CDNA was prepared using reverse transcriptase (reversetranscriptase) with poly A+primers on RNA. The design primers for IL-4was used in the PCR amplification (Table 1). GAPDH was used as internalstandard.

Experimental Example 3-2: Confirmation of IL-4 Secretion Inhibition byEnzyme-Linked Immune abSorbent Assay (ELISA)

The cells were treated with 0.1 pg/Me to 10 μg/Ml EC-18 for 1 hour andantigen was processed. After the incubation for 18 hours at 37° C., thecell was removed to obtain a supernatant. The quantification of rat IL-4which was present in the cell culture medium of RBL-2H3 cells was madeby carrying out enzyme-linked immunosorbent assay (ELISA) according tothe protocol indicated by the manufacturer using commerciallypurchasable monoclonal antibody (mAb) (BD Biosciences).

As a result, it was confirmed that in the RBL-2H3 cells treated with theEC-18, expression of IL-4 transcript was reduced in proportion to theconcentration of the EC-18 (FIG. 4) and expression of the secretion ofIL-4 induced by the antigen was reduced in accordance with the EC-18treatment in the RBL-2H3 cells (FIG. 5). In addition, it was confirmedthat the secretion of IL-4 expressed at each time was decreased in aEC-18 concentration-dependent manner, and at 10 μg/Ml concentration ofEC-18, secretion of IL-4 was inhibited by up to 60% or more.

Experimental Example 4: Antitumor Efficacy Analysis by EC-18 in CancerAnimal Models

The tumor growth inhibition was evaluated after the EC-18 of the testsubstance was orally administered to male nude mice to which RPMI 8226cells, derived from a human bone cancer cell lines were transplanted.The experiment included six groups: the negative control group; 500mg/kg dose of the test substance administration group; the combinationgroup to which 500 mg/kg of the test substance and 80 mg/kg dosepositive control substance (gemcitabine, Gemcitabine) are administrated;the combination group to which 500 mg/kg of test substance and 120 mg/kgdose of the positive control substance are administrated; 80 mg/kg doseof positive control group; and 120 mg/kg dose of positive control group.Each group consists of 10 mice. Olive oil an excipient was administeredto the negative control group. In test substance administration group,the administration of EC-18 to their stomachs was made once a day for 4weeks (a total of 28 administrations). In positive control group, theadministration of the positive control substance of Gemcitabine) to theabdominal cavity was made twice a week for 4 weeks (a total of 8administrations). During the observation period, common symptoms wereobserved once a day, weigh of animal and tumor volume (mm3) in animalswere measured twice a week. After the end of the observation period, thetumor was excised and the weight thereof (Tumor weight, g) was measured,the results are shown in FIGS. 6 and 7. As shown in FIGS. 6 and 7, inthe test substance administration group of 500 mg/kg dose, the volume ofthe tumors was significantly suppressed as compared to the negativecontrol group statistically, and the weight of the tumor was alsostatistically significant small compared to the negative control groupso that the growth of tumor was inhibited. Further, in the blood cancermodel mice, the photograph showing the change in size of the tumor afteradministration of the EC-18 and the positive control gemcitabine wasshown in FIG. 8. As shown in FIG. 8, it was confirmed that even when theEC-18 was administrated, the size of the tumor was reduced in similar tothe positive control.

From the above description, a person skilled in the art will appreciatethat the invention may be embodied in other specific forms withoutchanging the technical spirit or essential characteristics. In thisregard, the examples described above are intended to be illustrative inall respects and it should be understood as not limiting. The scope ofthe invention should be understood to include all ranges of the abovedetailed description and the appended claims, rather than the ranges ofthe specific examples, as well as all such modifications derived fromthose equivalents.

What is claimed:
 1. A method of modulating a cytokine in a subject comprising: administering to the subject a monoacetyldiacylglycerol of Formula I:

wherein R1 and R2 are independently a fatty acid group comprising 14 to 20 carbon atoms.
 2. The method of claim 1, wherein R1 and R2 are each independently selected from the group consisting of palmitoyl, oleoyl, linoleoyl, linolenoyl, stearoyl, myristoyl, and arachidonoyl.
 3. The method of claim 2, wherein R1 is palmitoyl.
 4. The method of claim 3, wherein R2 is linoleoyl.
 5. The method of claim 1, wherein the cytokine is IL-4, IL-13, or a combination thereof.
 6. The method of claim 1, wherein modulating the cytokine treats a blood cancer in a subject.
 7. The method of claim 6, wherein the blood cancer is selected from the group consisting of lymphoma, acute leukemia, chronic leukemia, and multiple myeloma.
 8. The method of claim 1, wherein the monoacetyldiacylglycerol reduces secretion of the cytokine from a cell in the subject, inhibits expression of the cytokine in a cell in the subject, or a combination thereof.
 9. The method of claim 8, wherein the cell is a cancer cell, a lymphocyte, a leukocyte, or a combination thereof.
 10. The method of claim 9, wherein the lymphocyte is a T cell, a tumor-infiltrating lymphocyte, or a combination thereof.
 11. The method of claim 9, wherein the leukocyte is a monocyte, an eosinophil, a mast cell, or a combination thereof.
 12. The method of claim 1, wherein the monoacetyldiacylglycerol of Formula I is in an amount of 0.001 to 50% by weight of a pharmaceutical composition.
 13. The method of claim 12, wherein the pharmaceutical composition is formulated for oral administration.
 14. The method of claim 13, wherein the pharmaceutical composition is a capsule, tablet, bolus, powder, or granule.
 15. The method of claim 1, wherein the subject is a human or a non-human animal.
 16. The method of claim 15, wherein the non-human animal is selected from the group consisting of a monkey, a dog, a cat, a rabbit, a guinea pig, a rat, a mouse, a cow, a sheep, a pig, and a goat.
 17. A method of reducing a cytokine in a subject comprising: administering to the subject a compound of Formula 2:

wherein the compound of Formula 2 reduces secretion of the cytokine from a cancer cell in the subject, inhibits expression of the cytokine in a cancer cell in the subject, or a combination thereof; and wherein the cytokine is IL-4. 